β-lactam derivatives as anti-inflammatory and antidegenerative agents

ABSTRACT

β-Lactam derivatives and analogs are found to be potent elastase inhibitors and thereby useful anti-inflammatory/antidegenerative agents.

BACKGROUND OF THE INVENTION

We have found that β-lactam derivatives and analogs are potent elastaseinhibitors and therefore are useful anti-inflammatory/anti-degenerativeagents.

Proteases from granulocytes and macrophages have been reported to beresponsible for the chronic tissue destruction mechanisms associatedwith inflammation, including rheumatoid arthritis and emphysema.Accordingly, specific and selective inhibitors of these proteases arecandidates for potent anti-inflammatory agents useful in the treatmentof inflammatory conditions resulting in connective tissue destruction,e.g. rheumatoid arthritis, emphysema, bronchial inflammation,osteoarthritis, spondylitis, lupus, psoriasis and acute respiratorydistress syndrome.

The role of proteases from granulocytes, leukocytes or macrophages arerelated to a rapid series of events which occur during the progressionof an inflammatory condition:

(1) There is a rapid production of prostaglandins (PG) and relatedcompounds synthesized from arachidonic acid. This PG synthesis has beenshown to be inhibited by aspirin-related nonsteroidal anti-inflammatoryagents including indomethacin and phenylbutazone. There is some evidencethat protease inhibitors prevent PG production;

(2) There is also a change in vascular permeability which causes aleakage of fluid into the inflamed site and the resulting edema isgenerally used as a marker for measuring the degree of inflammation.This process has been found to be induced by the proteolytic or peptidecleaving activity of proteases, especially those contained in theqranulocyte, and thereby can be inhibited by various synthetic proteaseinhibitors, for example, N-acyl benzisothiazolones and the respective1,1-dioxides. Morris Zimmerman et al., J. Biol. Chem., 255, 9848 (1980);and B. Ashe et al., J. Biol. Chem., 256, 11603

(1981);

(3) There is an appearance and/or presence of lymphoid cells, especiallymacrophages and polymorphonuclear leukocytes (PMN). It has been knownthat a variety of proteases are released from the macrophages and PMN,further indicating that the proteases do play an important role ininflammation.

In general, proteases are an important family of enzymes within thepeptide bond cleaving enzymes whose members are essential to a varietyof normal biological activities, such as digestion, formation anddissolution of blood clots, the formation of active forms of hormones,the immune reaction to foreign cells and organisms, etc., and inpathological conditions such as the degradation of structural proteinsat the articular cartilage/pannus junction in rheumatoid arthritis etc.

Elastase is one of the proteases. It is an enzyme capable of hydrolyzingthe connective tissue component elastin, a property not contained by thebulk of the proteases present in mammals. It acts on a protein'snonterminal bonds which are adjacent to an aliphatic amino acid.Neutrophil elastase is of particular interest because it has thebroadest spectrum of activity against natural connective tissuesubstrates. In particular, the elastase of the granulocyte is importantbecause, as described above, granulocytes participate in acuteinflammation and in acute exacerbation of chronic forms of inflammationwhich characterize many clinically important inflammatory diseases.

Proteases may be inactivated by inhibitors which block the active siteof the enzyme by binding tightly thereto. Naturally occurring proteaseinhibitors form part of the control or defense mechanisms that arecrucial to the well-being of an organism. Without these controlmechanisms, the proteases would destroy any protein within reach. Thenaturally occurring enzyme inhibitors have been shown to haveappropriate configurations which allow them to bind tightly to theenzyme. This configuration is part of the reason that inhibitors bind tothe enzyme so tightly (see Stroud, "A Family of Protein-CuttingProteins" Sci. Am. July 1974, pp. 74-88). For example, one of thenatural inhibitors, α₁ -Antitrypsin, is a glycoprotein contained inhuman serum that has a wide inhibitory spectrum covering, among otherenzymes, elastase both from the pancreas and the PMN. This inhibitor ishydrolyzed by the proteases to form a stable acyl enzyme in which theactive site is no longer available. Marked reduction in serum α₁-antitrypsin, either genetic or due to oxidants, has been associatedwith pulmonary emphysema which is a disease characterized by aprogressive loss of lung elasticity and resulting respiratorydifficulty. It has been reported that this loss of lung elasticity iscaused by the progressive, uncontrolled proteolysis or destruction ofthe structure of lung tissue by proteases such as elastase released fromleukocytes. J. C. Powers, TIBS, 211 (1976).

Rheumatoid arthritis is characterized by a progressive destruction ofarticular cartilage both on the free surface bordering the joint spaceand at the erosion front built up by synovial tissue toward thecartilage. This destruction process, in turn, is attributed to theprotein-cutting enzyme elastase which is a neutral protease present inhuman granulocytes. This conclusion has been supported by the followingobservations:

(1) Recent histochemical investigations showed the accumulation ofgranulocytes at the cartilage/pannus junction in rheumatoid arthritis;and

(2) a recent investigation of mechanical behavior of cartilage inresponse to attack by purified elastase demonstrated the directparticipation of granulocyte enzymes, especially elastase, in rheumatoidcartilage destruction. H. Menninger et al., in Biological Functions ofProteinases, H. Holzer and H. Tschesche, eds. Springer-Verlag, Berlin,Heidelberg, New York, 1979, pp. 196-206.

Accordingly, an object of this invention is to discover new proteaseinhibitors, especially elastase inhibitors, useful for controllingtissue damage and various inflammatory or degenerative conditionsmediated by proteases particularly elastase.

Another object of the present invention is to provide pharmaceuticalcompositions for administering the active β-lactam derivatives andanalogs as protease inhibitors.

Still a further object of this invention is to provide a method ofcontrolling inflammatory conditions by administering a sufficient amountof one or more of the active, β-lactam derivatives and analogs in amammalian species in need of such treatment.

DETAILED DESCRIPTION OF THE INVENTION A: Scope of the Present Invention

This invention relates to β-lactam derivatives especially penem andanalogs which are potent elastase inhibitors useful in the prevention,control and treatment of inflammatory conditions especially arthritisand emphysema.

Free acids of penem derivatives and analogs are known antibiotics whichhave been described in various patents. For example, U.S. Pat. Nos.4,260,618; 4,301,074; G.B. Pat. Nos. 201 3674 and 204 3649.

The compounds of the present invention are of formula: ##STR1## whereinQ is O or S;

n is 0, 1 or 2.

Preferably Q is O, n is 0 or 1.

For structure (I),

M is:

(1) --OR wherein R is as defined below;

(2) --SR;

(3) --S(O)R;

(4) --SO₂ R or --SO₃ R;

(5) --COOR;

(6) --OCOR or --OCOOR;

(7) phenyl;

(8) --CH₂ A wherein A represents

(a) hydrogen;

(b) loweralkyl especially C₁₋₆ alkyl;

(c) hydroxy;

(d) alkoxy;

(e) aryloxy;

(f) aralkyloxy;

(g) --SR;

(h) acylthio;

(i) acyloxy especially alkanoyloxy or arylcarbonyloxy such as acetoxy,benzyloxycarbonyloxy, benzoyloxy; and succinoyloxy; substituted orunsubstituted carbamoyl, thiocarbamoyl and N-alkyl or N,N-dialkylderivatives thereof;

(j) a quaternary ammonium group, for example, --NH₃, --NHE², or --NE³where E represents loweralkyl, aryl or aralkyl;

(k) unsubstituted or substituted amino or amido group especially --NH₂,--CONH₂ and N-alkyl or N,N-dialkyl derivatives thereof.

(1) --CH₂ N₃ ;

(m) halo;

(n) --CH₂ NH₂ ;

(o) --CH₂ NHCOOR

(p) --CH₂ NHCOR wherein R is:

(i) straight or branched alkyl chain having from 1 to 10 carbon atomsespecially methyl, ethyl, isopropyl, t-butyl, pentyl, or hexyl;

(ii) aryl having from 6 to 10 carbon atoms especially phenyl,substituted phenyl or naphthalene;

(iii) cycloalkyl having from 3 to 8 carbon atoms especially cyclopentyl,or cyclohexyl;

(iv) alkenyl having from 2 to 20 carbon atoms especially C₂₋₆ alkenylsuch as vinyl, allyl, or butenyl;

(v) cycloalkenyl having from 5 to 8 carbon atoms especialy cyclopentenylor cyclohexenyl;

(vi) alkynyl having from 2 to 20 carbon atoms especially for C₂₋₆alkynyl for example, ethynyl, propynyl or hexynyl;

(vii) aralkyl, alkaryl, aralkenyl, aralkynyl, alkenylaryl or alkynylarylwherein alkyl, aryl, alkenyl and alkynyl are as previously defined;

(viii) monoheteroaryl, di- or polyheteroaryl, or fused heteroarylcontaining from 1 to 3 of any one or more of the heteroatoms N, S or Oin each heteroaryl ring thereof, for example, pyridyl, pyrryl, thienyl,isothiazolyl, imidazolyl, pyrazinyl, pyrimidyl quinolyl, isoquinolyl,benzothienyl, tetrazolyl, isobenzofuryl pyrazolyl, indolyl, purinyl,carbozolyl, isoxazolyl and the like;

(ix) heteroarylalkyl such as 2-pyridylmethyl, 1-methyl-5-tetrazolyl,2-thienylmethyl and 3-isothiazolylethyl; or

(x) hydrogen;

the above groups (i)-(x) can be unsubstituted or can be substituted byradicals such as alkyl, alkoxy, halo such as fluoro, chloro, bromo oriodo, cyano, carboxy, sulfoamino, carbamoyl, alkyl or aminosulfonyl,azido, substituted amidino, amino, substituted amino such asmonoalkylamino and dialkylamino, haloalkyl, carboxyalkyl,carbamoylalkyl, N-substituted carbamoylalkyl, --NHCOOCH₃, guanidino,N-substituted guanidino, guanidinoalkyl, carboxamidino, N-substitutedcarboxamidino, tetrazolyl, and the like.

(9) hydrogen;

(10) trifluoromethyl;

(11) halo such as Cl or F;

(12) --CHO; ##STR2## wherein Q₁ and Q₂ independently are CN, orCOOR_(a).

Preferably, R is:

(a) H;

(b) C₁₋₆ alkyl;

(c) phenyl;

(d) --CH₂ CH₂ NH₂ ;

(e) --CH₂ CH₂ NH--COOCH₃ ;

(f) --CH₂ CH₂ OCH₃ ; ##STR3##

Thus, CH₂ A can be a halomethyl such as chloromethyl, bromomethyl orfluoromethyl.

When CH₂ A is a substituted hydroxy or substituted mercapto group, itcan be shown by the formula

    --CH.sub.2 ZR.sub.5

where Z is oxygen or sulfur, and R₅ is an acyl group; a straight chainor branched chain loweralkyl, alkenyl or alkynyl group; an aryl group;an aralkyl group; or a heterocyclic group such as heteroaryl,heterocycloalkyl e.g., 1,3-dioxacyclohex-4-yl, piperidino, morpholino,oxacyclopropyl, pyrrolidino, tetrazolo, benzothiazolo, imidazolidino,pyrazolidino, and piperazino; or heterocycloalkenyl such as pyrrolino,2-imidazolino, 3-pyrazolino or isoindolino. These groups can beunsubstituted or can be substituted by radicals such as alkyl, alkoxy,halo, cyano, carboxy, carbamoyl, azido,, sulfo, amino,, substitutedamino, haloalkyl, carboxyalkyl, carbamoylalkyl, N-substitutedcarbamoylalkyl, guanidino, N-substituted guanidino, guanidoalkyl,sulfamyl, substituted sulfamyl, and the like. Representative of the--CH₂ A groups are methoxymethyl, n-propoxymethyl, methylthiomethyl,acetoxymethyl, propionyloxymethyl, benzoyloxymethyl,(p-chlorobenzoyl)oxymethyl, succinoyloxymethyl,(p-methylbenzoyl)oxymethyl, pivaloyloxymethyl, D- orL-α-aminophenylacetyloxy, (1-adamantyl)-carboxymethyl,butanoyloxymethyl, carbamoyloxymethyl, (N-methylcarbamoyl)oxymethyl,(N-ethylcarbamoyl)oxymethyl, [N-(2-chloroethyl)carbamoyl]oxymethyl,(N-phenylcarbamoyl)oxymethyl, [N-(carboxymethyl)carbamoyl]oxymethyl,(N-p-sulfophenyl-carbamoyl)oxymethyl,(N-p-carboxymethylphenyl-carbamoyl)oxymethyl, methoxycarbonyloxymethyl,isobutanoyloxymethyl, cyclobutyl-carbonyloxymethyl, carbamoylthiomethyl,(ethoxythiocarbonyl)thiomethyl, (n-propoxythiocarbonyl)thiomethyl,(cyclopentanoxythiocarbonyl)thiomethyl, methylthiomethyl,N,N-diethylthiocarbamoylthiomethyl,N-methylpiperazinium-1-thiocarbonylthiomethyl,N,N-dimethylpiperazinium-1-thiocarbonylthiomethyl, 2-furoylthiomethyl,isothiouroniummethyl, (5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl,p-tolylsulfonylthiomethyl, 2-benzothiazolothiomethyl, mesyloxymethyl,1-methy-1,2,3,4-tetrazolyl-5-thiomethyl, tosyloxymethyl,sulfamoyloxymethyl, 1-naphthoyloxymethyl, 2-furylacetoxymethyl,cinnamoyloxymethyl, p-hydroxycinnamoyloxymethyl,p-sulfocinnamoyloxymethyl and 1R:2S-epoxypropylphosphonyloxymethyl.

Alternatively, when CH₂ A is hydroxymethyl, the penem derivative oranalog can also exist as the lactone which is formed by internalesterification with the adjacent carboxy group.

The substituent CH₂ A can also be a group of the general formula

    --CH.sub.2 Y.sub.1

wherein Y₁ represents amino or substitued amino including nitrogenheterocycles and substituted heterocyclic groups as described for R₅. Y₁may also be nitrogen which is part of the heterocyclic system as shownbelow.

Examples of such groups that might be mentioned are aminomethyl,acetamidomethyl, carbamoylaminomethyl, N,N-dimethylaminomethyl,N-(2-chloroethyl)aminomethyl, 5-cyano-triazol-1-yl-methyl,4-methoxy-carbonyltriazol-1-yl-methyl.

When A is amino the penem derivative or analog can also exist as thelactam formed by loss of water with the adjacent carboxy group.

Representative of the quaternary ammonium groups representing A thatmight be mentioned are pyridinium, 3-methylpyridinium,4-methylpyridinium, 3-chloropyridinium, 3-bromopyridinium,3-iodopyrinium, 4-carbamoylpyridinium,4-(N-hydroxymethylcarbamoyl)pyridinium,4-(N-carbomethoxycarbamoyl)pyridinium, 4-(N-cyanocarbamoyl)pyridinium,4-carboxymethylpyridinium, 4-hydroxymethylpyridinium,4-trifluoromethyl-pyridinium, quinolinium, picolinium and lutidinium.

When A is mercapto,i.e., --SR, it may be --SH, ##STR4## heteroarylcontaining nitrogen such as: ##STR5## alkylthio, arylthio, aralkylthioor heterocyclothio, wherein R_(a) represents C₁₋₆ alkyl or H.

The preferred groups representing A are

(a) hydrogen;

(b) C₁₋₆ alkyl

(c) phenyl;

(d) OR;

(e) halo;

(f) OCOR; ##STR6## (h) acylthio, i.e., --SCOR; (i) CH₂ NHCHO;

(j) --CH₂ N₃ ;

(k) CH₂ NH₂ ; or

(l) CH₂ OH.

The acyl group RCO-- can be a loweralkanoyl group of 2-6 carbon atomssuch as acetyl, --COC₂ H₅ or --COC₃ H₇, carbamoyl, or thiocarbamoyl andN-alkyl or N,N-dialkyl derivatives thereof. The alkyl group of theforegoing substituents contains 1-10 carbon atoms and may be furthersubstituted by radicals such as alkoxy, halo, amino, cyano, carboxy,sulfo, and the like.

More preferably, A is ##STR7## (b) C₁₋₃ alkoxy especially methoxy,ethoxy or i- or n-propyloxy: (c) phenyl;

(d) hydrogen;

(e) hydroxy; or

(f) SR or ##STR8##

For structure (II),

M can only be ═O, ═S, or ═CHA;

R₁ is

(a) nitrogen bonded group including R'NH-- wherein R' is as definedbelow;

(b) hydrogen;

(c) hydroxy;

(d) mercapto;

(e) substituted oxy;

(f) substituted thio;

(g) hydrocarbyl or substituted hydrocarbyl group;

(h) cyano;

(i) carbonyl or thiocarbonyl containing substituents bonded by saidcarbonyl or thiocarbonyl radical;

(j) halo;

(k) phosphono or a substituted phosphono group; or

(l) hydroxyalkyl especially hydroxy-C₁₋₆ alkyl such as CH₃ CH(OH)--;

(m) alkoxycarbonyloxyalkyl especially C₁₋₆ alkoxycarbonyloxy C₁₋₆ alkylsuch as CH₃ CH(OCOOt--Bu)--;

(n) benzoxycarbonyloxy-C₁₋₆ alkyl, e.g., CH₃ CH(OCOOCH₂ C₆ H₅)--;

(o) phenoxycarbonyloxy-C₁₋₆ alkyl such as CH₃ CH(OCOOC₆ H₅)--; or

(p) alkoxycarbonylalkyl.

When R₁ is R'NH--, R' represents a substituted or unsubstitutedaliphatic, aromatic or heterocyclic, araliphatic orheterocyclylaliphatic carboxylic acid radical or a carbothioic acidradical such as the acyl radicals of the known cephalosporins andpenicillins. These acyl radicals can be represented by the generalformula ##STR9## where R₂ is a radical of the group defined below, m andn represent 0-4 and R₃ represents R" or ZR", which are also definedbelow.

One group of the acyl radicals, i.e., when m and n are both 0 and R₃ isR", can be represented by the general formula ##STR10## wherein R" is:(a) straight or branched chain alkyl having from 1 to 20 carbon atomsespecially methyl, trifluoromethyl, ethyl, isopropyl, t-butyl, pentyl orhexyl;

(b) aryl having from 6 to 10 carbon atoms especially phenyl, substitutedphenyl or naphthalene;

(c) cycloalkyl having from 3 to 8 carbon atoms especially cyclopentyl,or cyclohexyl;

(d) alkenyl having from 2 to 20 carbon atoms especially C₂₋₆ alkenylsuch as vinyl, allyl, or butenyl;

cycloalkenyl having from 5 to 8 carbon atoms especialy cyclopentenyl orcyclohexenyl;

(f) alkynyl having from 2 to 20 carbon atoms especially C₂₋₆ alkynyl forexample, ethynyl, propynyl or hexynyl;

(g) alkoxy having from 1 to 10 carbon atoms especially C₁₋₃ alkoxy suchas methoxy, ethoxy or n-propoxy or i-propoxy;

(h) aralkyl, alkaryl, aralkenyl, aralkynyl, alkenylaryl or alkynylarylwherein alkyl, aryl, alkenyl and alkynyl are as previously defined;

(i) monoheteroaryl, di- or polyheteroaryl, or fused heteroarylcontaining from 1 to 3 of any one or more of the heteroatoms N, S or Oin each heteroaryl ring thereof, for example, pyridyl, pyrryl, thienyl,isothiazolyl, imidazolyl, pyrazinyl, pyrimidyl quinolyl, isoquinolyl,benzothienyl, isobenzofuryl pyrazolyl, indolyl, purinyl, carbozolyl,isoxazolyl and the like;

(j) heteroarylalkyl such as 2-pyridylmethyl, 2-thienylmethyl and3-isothiazolylethyl; or

(k) hydrogen.

The above groups (a)-(b) can be unsubstituted or can be substituted byradicals such as alkyl, alkoxy, halo such as fluoro, chloro, bromo oriodo, cyano, carboxy, sulfoamino, carbamoyl, sulfonyl, azido, amino,substituted amino such as monoalkylamino and dialkylamino, haloalkyl,carboxyalkyl, carbamoylalkyl, N-substituted carbamoylalkyl, guanidino,N-substituted guanidino, guanidinoalkyl, and the like. Representativeexamples of such acyl groups that might be mentioned are those whereinR" is methoxy, ethoxy, benzyl, p-hydroxybenzyl, 3- or 4-nitrobenzyl,p-aminobenzyl, o-aminobenzyl, m-aminobenzyl, o-sulfobenzyl,p-carboxymethylbenzyl, p-carbamoylmethylbenzyl, m-fluorobenzyl,m-bromobenzyl, p-chlorobenzyl, p-methoxybenzyl, p-aminomethylbenzyl,hydrogen, methyl, ethyl, cyanomethyl, 2-pentenyl, n-amyl, n-heptyl,phenethyl, difluoromethyl, trifluoromethyl, dichloromethyl,dibromoethyl, 1-(3-methylimidazolyl)-methyl, 2- or3-(5-carboxymethylthienyl)methyl, 2- or 3-(4-carbamoylthienyl)methyl, 2-or 3-(5-methylthienyl)methyl, 2- or 3-(5-methoxythienyl)methyl, 2- or3-(4-chlorothienyl)methyl, 2- or 3-(5-sulfothienyl)methyl, 2- or3-(5-carboxythienyl)methyl, 3-(1,2,5-thiadiazolyl)methyl,3-(4-methoxy-1,2,5-thiadiazolyl)methyl, 2-furylmethyl,2-(5-nitrofuryl)methyl, 3-furylmethyl, 2-thienylmethyl, andtetrazolylmethyl. The term "sulfo" represents mercapto or thio, sulfinyland sulfonyl.

The acyl group can also be a radical of the formula ##STR11## wherein nis 0-4, Z represents oxygen, sulfur or nitrogen, and R" is defined asabove. Representative members of the substituent

    --(CH.sub.2).sub.n ZR"

are allylthiomethyl, allylaminomethyl, phenylthiomethyl,butylmercaptomethyl, α-chlorocrotylmercaptomethyl, phenoxymethyl,phenylaminomethyl, phenoxyethyl, phenoxybutyl, phenoxybenzyl,,diphenoxymethyl, dimethylmethoxymethyl, dimethylbutoxymethyl,dimethylphenoxymethyl, 4-guanidinophenoxymethyl, 4-pyridylthiomethyl,p-(carboxymethyl)phenoxymethyl, p-(carboxymethyl)phenylthiomethyl,2-thiazolylthiomethyl, p-(sulfo)phenoxymethyl,p-(sulfo)phenylthiomethyl, p-(carboxy)phenoxymethyl,p-(carboxy)phenylthiomethyl, p-(carboxymethyl)phenoxymethyl,p-carboxymethyl)phenylthiomethyl, 2-pyrimidinylthiomethyl,phenethylthiomethyl, 1-(5,,6,7,8-tetrahydronaphthyl)oxomethyl,6,8-bis(methylthio)octanoyl.

Furthermore, the acyl group can be a radical of the formula ##STR12##wherein R" is defined as above and R"' is a radical such as amino,hydroxy, azido, carbamoyl, guanidino, alkanoyloxy, halo, sulfamino,tetrazolyl, sulfo, carboxy, carbalkoxy, and the like. Representativemembers of the substituent ##STR13## are α-aminobenzyl,α-amino-2-thienyl, α-methylaminobenzyl, α-amino-γ-methylmercaptopropyl,α-amino-3 or 4-chlorobenzyl, α-amino-3 or 4-hydroxybenzyl,α-amino-2,4-dichlorobenzyl, α-amino-3,4-dichlorobenzyl,D(-)-α-hydroxybenzyl, α-carboxybenzyl, α-amino-3-thienyl,α-amino-2-thienyl, D(-)-α-amino-3-chloro-4-hydroxybenzyl,D(-)-α-amino-3-thienyl, 1-aminocyclohexyl, α-(5-tetrazolyl)benzyl,α-sulfaminobenzyl, α-sulfamino-3-thienyl, α-(N-methylsulfamino)benzyl,D(-)-α-guanidino-2-thienyl, D(-)-α-guanidinobenzyl,α-guanylureidobenzyl, α-hydroxybenzyl, α-azidobenzyl, α-fluorobenzyl,4-(5-methoxy-1,3-oxadiazolyl)aminomethyl,4-(5-methoxy-1,3-oxadiazolyl)-hydroxymethyl,4-(5-methoxy-1,3-oxadiazolyl)-carboxymethyl,4-(5-methoxy-1,3-sulfadiazolyl)-aminomethyl,4-(5-methoxy-1,3-sulfadiazolyl)-hydroxymethyl,4-(5-methoxy-1,3-sulfadiazolyl)-carboxymethyl,2-(5-chlorothienyl)-aminomethyl, 2-(5-chlorothienyl)hydroxymethyl,2-(5-chlorothienyl)-carboxymethyl, 3-(1,2-thiazolyl)aminomethyl,3-(1,2-thiazolyl)hydroxymethyl, 3-(1,2-thiazolyl)-carboxymethyl,2-(1,4-thiazolyl)aminomethyl, 2-(1,4-thiazolyl)-hydroxymethyl,2-(1,4-thiazolyl)-carboxymethyl, 2-benzothienylaminomethyl,2-benzothienylhydroxymethyl, 2-benzothienylcarboxymethyl,2-azidooctyl-3-phenyl-3-azidomethyl, α-sulfobenzyl, andα-phosphonobenzyl.

Alternatively, the group ##STR14## can be an unsubstituted orsubstituted alkyl or aryl sulfonamido group wherein R' is ; C₁₋₆alkyl-SO₂ ; CF₃ SO₂ ; C₆ H₅ SO₂ ; or C₆ H₅ CH₂ SO₂. For example,phenylsulfonamido, ethylsulfonamido, trifluoromethane sulfonamido,benzylsulfonamido, 2,5-dimethylsulfonamido, 4-chlorophenylsulfonamido,4-methoxyphenylsulfonamido, or an unsubstituted or substituted alkyl oraryl sulfonylalkylamino group wherein R' is CH₃ SO₂ CH(CH₃)-- or C₆ H₅SO₂ CH₂, and the like.

Preferably, R' is:

(1) hydrogen; ##STR15## R² represents: (a) hydrogen;

(b) methyl or substituted methyl such as trifluoromethyl, cyanomethyl amethoxymethyl;

(c) thienyl;

(d) phenyl; or

(e) mono- and disubstituted phenyl and thienyl wherein the substituentsare selected from the group consisting of chloro, bromo, fluoro, nitro,loweralkyl, and loweralkoxy;

n is 0 or 1; or ##STR16## X₁ is oxygen or sulfur; R² and n are aspreviously defined.

Even more preferably, R' is ##STR17## being selected from the groupconsisting of: (1) trifluoromethyl;

(2) methyl;

(3) methoxy;

(4) hydrogen;

(5) benzyl;

(6) phenyl;

(7) 2-thienylmethyl;

(8) phenylthiomethyl;

(9) phenoxymethyl;

(10) benzyloxy, or

(11) NCCH₂ SCH₂.

The oxy or thio substituent represented by R₁ can be a substitutedhydroxy or mercapto group such as --XR'₁ l wherein X is oxygen or sulfurand R'₁ is a hydrocarbyl group, preferably a straight or branchedloweralkyl group of 1-6 carbon atoms, a straight or branched chainloweralkenyl or loweralkynyl group of 3-6 carbon atoms, a monocyclicaryl group such as phenyl, furyl, pyrryl and pyridyl, or an aralkylgroup such as benzyl. These alkyl, alkenyl, alkynyl, aryl or aralkylgroups can be substituted with groups such as hydroxy, halo, nitro,amino, carboxy, thio, and the like. Other specific substituentsrepresented by R₁ that might be mentioned are groups of the formula--OAc, --SAc, --SO₃ H, --SO₂ NH₂, --OCD₃, --SO₂ R₂, --SO₂ NR₃ R₄,--OCOOR₂, --SOR₂, --OCOSR₂, --OCONR₃ R₄, and the like wherein Acrepresents an acyl group such as a formyl or loweralkanoyl, R₃ and R₄represent hydrogen, loweralkyl, acyl and loweralkoxy, and R₂ representsloweralkyl, haloloweralkyl, aryl, aralkyl and substituted derivatives ofsuch groups.

When R₁ is hydrocarbyl it can be straight or branched loweralkyl,straight or branched lower-alkenyl, loweralkynyl, aralkyl, cycloalkyl, amonocyclic aryl group, or a monocyclic heterocyclic group which can alsobe substituted with one or more groups such as halo, hydroxy, alkoxy,amino, nitro, sulfonyl, sulfamoyl, acyloxy, carbamoyloxy, carboxy,carboxamido and N-substituted carboxamido. Representative examples ofsuch groups are C₁₋₆ alkyl such as methyl, trifluoromethyl, ethyl,n-propyl, isopropyl, t-butyl; C₂₋₆ alkenyl especially allyl, α-butenyl;C₂₋₆ alkynyl such as ethynyl and methylethynyl; loweraralkyl such asbenzyl, p-methoxybenzyl, phenethyl; phenyl, p-aminophenyl; cyclopropyl,cyclopentyl and 4-hydroxycyclohexyl;

R₁ may also represent cyano or a group of the general formula ##STR18##wherein X' is oxygen or sulfur, and R" is hydrogen, halo, hydroxy,mercapto, amino, substituted amino, alkyl, aryl, aralkyl, aralkoxy suchas benzyloxy, alkoxy or aryloxy such as phenoxy, pyrroloxy, furyloxy,and thienyloxy, alkylthio or arylthio. Examples of these substituentsare --COOH, --CSSH, --COR₂, --COOR₂, --COSR₂, --CSSR₂, --CONH₂, --CSNH₂,--CSR₂, --CONHR₂, --CSNH, --CONR₃ R₄ and --CSNR₃ R₄ wherein R₂represents a straight or branched chain alkyl group of 1-6 carbon atomsand R₃ and R₄ represent hydrogen or R₂ ;

Furthermore, R₁ represents a nitrogen bonded group such as amino,substituted amino groups, nitro, azido, nitroso, isocyanato,isothiocyanato and hydroxyamino. Specific examples of nitrogen bondedgroups that might be mentioned are --N₃, --NH₂, --NHR₂, NR₂ R₃, whereinR₂ represents a straight or branched chain loweralkyl group of 1 to 6carbon atoms, R₃ represents R₂ or hydrogen, and n represents the integer1 or 2.

Finally, the substituent R₁ represents phosphono or a metal or ammoniumsalt thereof, or a substituted phosphono group of the formula: ##STR19##where Y' and Z' are the same or different and represent ##STR20## R₂represents hydrogen or a hydrocarbyl radical, R₃ and R₄ representhydrogen, hydrocarbyl, alkoxy or an acyl radical, and X' representsoxygen or sulfur.

Preferably, R₁ is

(1) R'NH wherein R' is

(a) hydrogen;

(b) CF₃ CO or CH₃ OCO;

(c) C₁₋₆ alkyl SO₂ ;

(d) C₆ H₅ CH₂ SO₂ ;

(2) C₁₋₆ alkoxycarbonyloxyC₁₋₆ alkyl;

(3) benzoxycarbonyloxyC₁₋₆ alkyl;

(4) hydroxyalkyl;

(5) OR where R is as previously defined;

(6) SR;

(7) hydrogen;

(8) C₁₋₆ alkyl;

(9) C₁₋₆ alkoxycarbonyl C₁₋₆ alkyl; or

(10) phenoxycarbonyloxy C₁₋₆ alkyl.

Even more preferably, R₁ is

(1) C₁₋₃ alkyl especially ethyl and isopropyl;

(2) hydroxy C₁₋₃ alkyl especially CH₃ CH(OH)--;

(3) OR where R is

(a) C₁₋₆ alkyl especially methyl, ethyl, i-propyl; or ##STR21## where Rrepresents hydrogen, C₁₋₆ alkyl, phenyl, substituted or unsubstitutedbenzyl, or C₁₋₆ alkylamino such as CH₃ NH--, C₂ H₅ NH--;

(4) benzoxycarbonyloxy C₁₋₃ alkyl, e.g., CH₃ CH(OCOOCH₂ C₆ H₅)--;

(5) hydrogen; or

(6) C₁₋₆ alkoxycarbonyl C₁₋₃ alkyl.

B represents OB₁, or NB₂ B₃ wherein B₁ and B₂ independently are:

(a) straight or branched chain alkyl having from 1 to 20 carbon atoms,ethyl, isopropyl, t-butyl, pentyl or hexyl;

(b) aryl having from 6 to 10 carbon atoms;

(c) cycloalkyl having from 3 to 8 carbon atoms;

(d) alkenyl having from 2 to 20 carbon atoms;

(e) cycloalkenyl having from 5 to 8 carbon atoms;

(f) alkynyl having from 2 to 20 carbon atoms;

(g) aralkyl, alkaryl, aralkenyl, aralkynyl, alkenylaryl or alkynylarylwherein alkyl, aryl, alkenyl and alkynyl are as previously defined;

(h) loweralkenylalkyl;

(i) alkanoylalkyl;

(j) alkanoyloxyalkyl;

(k) alkoxyalkyl;

(l) a heterocyclic group including heterocyclic alkyl or heterocyclicalkenyl.

The above groups (a)-(l) can be unsubstituted or can be substituted byradicals such as alkyl, hydroxy, alkoxy, halo, nitro, mercapto, amino,substituted amino, cyano, carboxy, sulfoamino, carbamoyl, carbamoyloxy,alkyl or amino sulfonyl, alkyl or amino sulfinyl, sulfamoyl, azido,amino, substituted amino, carboxamido or N-substituted carboxamido; and

B₃ is hydrogen or B₁. Representative examples of such groups are C₁₋₆alkyl especially methyl, ethyl or t-butyl, allyl, 3-butenyl,methoxyethyl, benzyl, p-carbomethoxybenzyl, m-carbomethoxybenzyl,p-sulfonylbenzyl, m-fluorobenzyl, o,p-dinitrobenzyl, o,p-dichlorobenzyl,p-methylbenzyl, m-methoxybenzyl, o-methylthiobenzyl, benzhydryl, CH₂ CH₂CH₂ COOCH₃, --CH₂ COOC₂ H₅, and the like.

Preferably B₁ and B₂ independently are substituted or unsubstituted

(1) aralkyl;

(2) aryl;

(3) straight or branched loweralkyl;

(4) straight or branched loweralkenyl;

(5) cycloalkyl;

(6) alkanoyloxyloweralkyl;

(7) alkanoylloweralkyl;

(8) alkoxyloweralkyl; or

(9) haloalkyl; and

B₃ is H or B₁.

Even more preferably, B₁ and B₂ independently are substituted orunsubstituted

(1) benzyl such as p-nitrobenzyl;

(2) ethyl;

(3) t-butyl;

(4) --CH₂ CH₂ CH═CH₂ or CH₂ --CH═C(CH₃)₂ ;

(5) --CH₂ CH₂ CH₂ COOt-Bu;

(6) alkanoyloxymethyl; or

(7) alkanoylmethyl; and

B₃ is H or B₁.

It should be noted that the β-lactam derivatives can be active elastaseinhibitors in the form of an optical isomer, for example, l-, d- ordl-forms.

B. Preparation of The Compounds Within The Scope of the Invention

The penem derivatives and analogs of formula (I) or (II) where OB₁ isother than hydroxy can be prepared from the corresponding acid accordingto conventional methods of esterification. For example,

(1) A compound of formula (I) or (II) wherein B is OH is treated with alower alkanol, a substituted or unsubstituted benzyl alcohol, or asubstituted or unsubstituted benzhydrol (diphenylmethanol) in thepresence of a catalyst such as sulfuric acid, hydrochloric acid and anyone or a combination of the acid illustrated below in Table I at fromabout 0° to about 150° C. with or without refluxing until theesterification is substantially complete. Optionally, a solvent may beused to facilitate the reaction. The common solvents used are benzene,toluene, xylene, sulfolane-xylene, diethylether, tetrahydrofuran,1,2-dimethoxyethane, dioxane and the like;

(2) A compound of formula (I) or (II) is converted to an acid halidesuch as acid chloride or bromide via treatment with a halogenating agentsuch as thionyl chloride, phosphorus penta- or oxychloride followed byreaction with an appropriate alcohol; and

(3) Other methods such as alkylation of carboxylate salts (e.g., K⁺,Na⁺, Ca⁺⁺, Ag⁺, Cu⁺ tetralkylammonium-R₄ N⁺, and Hg⁺⁺ salts) of formula(I) or (II) with alkyl halides, for example, benzylchloride, benzyhydrylchloride; reaction with alkyl isoureas; treatment with diazomethane ordiazophenylmethane (C₆ H₅ CHN₂); alcoholysis of anhydride derived fromthe cephalosporin acid corresponding to formula (I) or (II);transesterification with t-butyl esters or i-propenyl acetate and thelike may also be used. These methods are disclosed in Saul Patai,editor, The Chemistry of Functional Groups, Supplement B, The Chemistryof Acid Derivatives, pp. 411-436, John Wiley & Sons, Chichester-NewYork-Brisbane-Toronto, 1979, and are incorporated herein by reference.

TABLE I Catalysts for Esterification

(1) Hydrochloric acid or hydrobromic acid

(2) Sulfuric acid

(3) C₁₋₃ alkanoic acid e.g. acetic acid

(4) Phosphoric acid

(5) Trifluoroacetic acid or anhydride

(6) Trichloroacetic acid

(7) p-Toluenesulfonic acid or other arylsulfonic acids

(8) Acidic ion-exchange resins with calcium sulfate

(9) Polymer-protected aluminum chloride, e.g., a complex betweenanhydrous aluminum chloride and polystyrene-divinyl benzene copolymerdiphenylphosphitepyridine

(10) A Lewis acid such as boron trifluoride

(11) Aromatic sulfonylchloride-pyridine, e.g., p-toluenesulfonylchloride

(12) triphenylphosphine ditriflate

(13) dicyclohexylcarbodiimide (DCCD)

(14) β-trichloromethyl-β-pro-piolactone

(15) N,N'-carbonyldimidazole

(16) triphenylphosphinediethylazodicarbonylate

(17) 6-chlorobenzensulfonyloxybenzotriazole

(18) 1-methyl-2-halopyridinium iodide-tertiary amine (e.g.,triethylamine).

The starting penem carboxylic acids are either known or can be preparedfrom known procedures as illustrated in the following table:

                                      TABLE II                                    __________________________________________________________________________    Synthesis of Penem Carboxylic Acids                                           Conversion                             Reference                              __________________________________________________________________________     ##STR22##                             GB 2,042,508A                           ##STR23##                             U.S. Pat. No. 4,169,833                 ##STR24##                             U.S. Pat. No. 4,301,074,                ##STR25##                             G.B. 2,043,639                          ##STR26##                             G.B. 2,013,674                          ##STR27##                             M. Foglio et al., Heterocycles,                                               16, 1919 (1981)                         ##STR28##                             I. Ernest, J. Gostali and R. B.                                               Woodward;                                                                     J. Am. Chem.                                                                  Soc., 101, 6301 (1979)                 __________________________________________________________________________

C. Utility of the Compounds Within the Scope of the Invention

This invention also relates to a method of treatment for patients (ormammalian animals raised in the dairy, meat, or fur industries or aspets) suffering from inflammation or pain. More specifically, it relatesto a method of treatment involving the administration of a compound offormula (I) or (II) as the active constituent.

For the treatment of inflammation and pain a compound of formula (I) or(II) may be administered orally, topically, parenterally, by inhalationspray or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.The term parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques. In addition to the treatment of warm-blooded animals such ashorses, cattle, sheep, dogs, cats, etc., the compounds of the inventionare effective in the treatment of humans.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparation. Tablets containing the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients may alsobe manufactured by known methods. The excipients used may be forexample, (1) inert diluents such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; (2) granulating anddisintegrating agents such as corn starch, or alginic acid; (3) bindingagents such as starch, gelatin or acacia, and (4) lubricating agentssuch as magnesium stearate, stearic acid or talc. The tablets may beuncoated or they may be coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. They may also be coated by the techniques described in theU.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for controlled release.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin. They may also be in the form of soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients may be

(1) suspending agents such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia;

(2) dispersing or wetting agents which may be

(a) a naturally-occurring phosphatide such as lecithin,

(b) a condensation product of an alkylene oxide with a fatty acid, forexample, polyoxyethylene stearate,

(c) a condensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethyleneoxycetanol,

(d) a condensation product of ethylene oxide with a partial esterderived from a fatty acid and a hexitol such as polyoxyethylene sorbitolmonooleate, or

(e) a condensation product of ethylene oxide with a partial esterderived from a fatty acid and a hexitol anhydride, for examplepolyoxyethylene sorbitan monooleate.

The aqueous suspensions may also contain one or more preservatives, forexample, ethyl or n-propyl p-hydroxybenzoate; one or more coloringagents; one or more flavoring agents; and one or more sweetening agentssuch as sucrose or saccharin.

Oily suspension may be formulated by suspending the active ingredient ina vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents may beadded to provide a palatable oral preparation. These compositions may bepreserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules are suitable for the preparation of anaqueous suspension. They provide the active ingredient in admixture witha dispersing or wetting agent, a suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example, those sweetening, flavoring and coloring agentsdescribed above may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil such asolive oil or arachis oils, or a mineral oil such as liquid paraffin or amixture thereof. Suitable emulsifying agents may be (1)naturally-occurring gums such as gum acacia and gum tragacanth, (2)naturally-occurring phosphatides such as soy bean and lecithin, (3)esters or partial esters derived from fatty acids and hexitolanhydrides, for example, sorbitan monooleate, (4) condensation productsof said partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavorng agents.

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative and flavoringand coloring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isototonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

A compound of (I) or (II) may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the anti-inflammatory agents are employed.

Dosage levels of the order from about 1 mg to about 100 mg per kilogramof body weight per day are useful in the treatment of theabove-indicated conditions (from about 50 mg to about 5 gms. per patientper day). For example, inflammation is effectively treated andanti-pyretic and analgesic activity manifested by the administrationfrom about 2.5 to about 75 mg of the compound per kilogram of bodyweight per day (about 75 mg to about 3.75 gms per patient per day).

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may containfrom 5 mg to 5 gm of active agent compounded with an appropriate andconvenient amount of carrier material which may vary from about 5 toabout 95 percent of the total composition. Dosage unit forms willgenerally contain between from about 25 mg to about 500 mg of activeingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

D. Biological Evidence in Support of Utility of the Invention

This invention also relates to a method of treating inflammation inpatients using a compound of Formula (I) or (II), particularly anespecially preferred compound as the active constituent.

It has been found that the compounds of Formula (I) or (II) haveanti-inflammatory/anti-degeneration activity as shown below in TablesIII and IV by the effective inhibition of the proteolytic function ofhuman granulocyte elastase.

                                      TABLE III                                   __________________________________________________________________________     ##STR29##                                                                    R.sub.1       n M          B          IC.sub.50 (μg/ml)                    __________________________________________________________________________    H             0 CH.sub.3   OCH.sub.2 C.sub.6 H.sub.5                                                                4                                       H             1 CH.sub.3   OCH.sub.2 C.sub.6 H.sub.5                                                                15                                      (CH.sub.3).sub.2 CH                                                                         0 CH.sub.2 C.sub.6 H.sub.5                                                                 OCH.sub.2 C.sub.6 H.sub.5                                                                0.6                                     (CH.sub.3).sub.2 CH                                                                         0 C.sub.6 H.sub.5                                                                          OCH.sub.2 C.sub.6 H.sub.5                                                                1.0                                     CH.sub.3 O    0 CH.sub.2 OCOCH.sub.3                                                                     OCH.sub.2 C.sub.6 H.sub.5                                                                4.0                                     CH.sub.3 CH(OH)                                                                             0 SC.sub.2 H.sub.5                                                                         OCH.sub.2 (p-NO.sub.2C.sub.6 H.sub.5)                                                    2.0                                     CH.sub.3 CH(OCOOCH.sub.2 C.sub.6 H.sub.5)                                                   0 SC.sub.2 H.sub.5                                                                         OCH.sub.2 (p-NO.sub.2C.sub.6 H.sub.5)                                                    0.2                                     (CH.sub.3).sub.2 CH                                                                         0 SC.sub.2 H.sub. 5                                                                        OCH.sub.2 C.sub.6 H.sub.5                                                                3.0                                     (CH.sub.3).sub.2 CH                                                                         0                                                                                ##STR30## OCH.sub.2 C.sub.6 H.sub.5                                                                0.7                                     H             0 OC.sub.2 H.sub.5                                                                         OCH.sub.2 C.sub.6 H.sub.5                                                                2.0                                     C.sub.2 H.sub.5                                                                             0 C.sub.6 H.sub.5                                                                          OCH.sub.2 C.sub.6 H.sub.5                                                                0.3                                     __________________________________________________________________________

                  TABLE IV                                                        ______________________________________                                         ##STR31##                                                                    B.sub.1         IC.sub.50 (μg/ml)                                          ______________________________________                                        H               inactive                                                      (clavulanic acid)                                                             CH.sub.2 C.sub.6 H.sub.5                                                                      3                                                             ______________________________________                                    

PROTOCOL Enzyme Assays for the Inhibition of Human PolymorphonuclearLeukocyte Elastase Via Hydrolysis ofN-t-Boc-alanyl-alanyl-prolylalanine-p-nitroanilide

Reagents:

0.05M TES (N-tris[hydroxymethyl]methyl-2-amino-ethanesulfonic acid)buffer, pH 7.5.

0.2 mM N-t-Boc-alanyl-alanyl-prolyl-alanine-p-nitroanilide (Boc-AAPAN).

To prepare substrate, the solid (m.w. 550) was first dissolved in 10.0ml DMSO. Buffer at pH 7.5 was then added to a final volume of 100 ml.

Crude extract of human polymorphonuclear leukocytes (PMN) containingelastase activity.

Inhibitors (β-lactam derivatives) to be tested dissolved in DMSO justbefore use.

Assay Procedure:

To 1.0 ml of 0.2 mM Boc-AAPAN in a cuvette, 0.01-0.1 ml of DMSO with orwithout inhibitor was added. After mixing, a measurement was taken at410 mμ to detect any spontaneous hydrolysis due to presence of testcompound. 0.05 Milliliters of PMN extract was then added and the ΔOD/minat 410 mμ was measured and recorded. Beckman model 35 spectrophotometerwas used.

Results:

Results were reported as ED₅₀, i.e., effective dosage in micrograms permilliliter (μg/ml) for 50% inhibition of the enzyme activity 2 minutesafter zero time.

Comments:

The elastase activity in the crude PMN extract may vary from onepreparation to another. A control of each new batch is run, and thevolume added in the assay procedure is adjusted according to activity.

The following examples illustrate the invention.

EXAMPLE 1 Benzyl 2-phenylpenem-3-carboxylate Step A: Preparation of4-(Triphenylmethylthio)azetidin-2-one

Sodium hydride (60%) (330 mg) was washed 3 times with hexane, suspendedin dimethylformamide (DMF) (10 ml) and cooled to 0° C. under nitrogen.Triphenylmethyl mercaptan (2.4 g) in DMF (5 ml) was added dropwise over10 minutes. The mixture was stirred for another 15 minutes before4-(acetoxy)azetidin-2-one (1.0 g) in DMF (10 ml) was added dropwise over15 minutes. After 15 minutes at 0° C., the reaction was quenched intocold, saturated ammonium chloride solution and extracted with ether(3×). Each ether extract was washed (brine), dried (Na₂ SO₄) andevaporated. The residue was purified by flash chromatography with asolvent gradient of 25 to 40% ethyl acetate/hexane to afford4-(triphenylmethylthio)azetidin-2-one (2.5 g) as a foam.

NMR δ(CDCl₃): 2.80 (1H, dm, J=15 Hz), 3.24 (1H, ddd, J=15 Hz, J=5 Hz,J=1.5 Hz), 4.3-4.5 (2H, dd over am, J=5 Hz, J=2 Hz), 6.9-7.6 (15H, m).

Step B: Preparation of4-(Triphenylmethylthio)-1-(benzyloxycarbonylhydroxymethyl)azetidin-2-one

A solution of 4-(triphenylthio)azetidin-2-one (3.45 g) and benzylglyoxylate (2.0 g) (prepared by ozonolysis of dibenzyl fumarate) inacetonitrile (30 ml) was stirred in the presence of 4 Angstrom sieves (5g) at room temperature for 4 days. The reaction was filtered andevaporated to a yellow oil. Preparative liquid chromatography (using 40%ethyl acetate/hexane) gave4-(triphenylmethylthio)-1-(benzyloxycarbonylhydroxymethyl)azetidin-2-one(5.0 g) as two separable diastereomers.

Higher R_(f) isomer (40% EtOAc/Hex): NMR δ(CDCl₃): 2.26 (2H, br. t, J=3Hz), 3.74 (1H, br. d, J=9 Hz), 4.31 (1H, br. t, J=3 Hz), 4.96 (1H, br.d, J=9 Hz), 5.20 (2H, s), 7.0-7.6 (20H, m). Lower R_(f) isomer (40%EtOAc/Hex):

NMR δ(CDCl₃): 2.73 (2H, br. d, J=3.5 Hz), 3.70 (1H, br. d, J=7 Hz), 4.43(1H, br. t, J=3.5 Hz), 5.17 (1H, br. d, J=7 Hz), 5.2 (2H, s), 7.0-7.5(20H, m).

Step C: Preparation of4-(Triphenylmethylthio)-1-(benzyloxycarbonylcarbonylchloromethyl)azetidin-2-one

To a solution of4-(triphenylmethylthio)-1-(benzyloxycarbonylhydroxymethyl)azetidin-2-one(1.0 g) and pyridine (240 μl) in tetrahydrofuran (THF) (15 ml) at 0° C.under nitrogen was added slowly thionyl chloride (180 μl ). After 15minutes the precipitate was collected by filtration and the filtrateevaporated. The residue was rapidly eluted through a short plug ofsilica gel using a 15 to 30% ethyl acetate/hexane solvent gradient toafford4-(triphenylmethylthio)-1-(benzyloxycarbonylchloromethyl)azetidin-2-one(900 mg) as a 4:3 mixture of diastereomers.

NMR δ(CDCl₃): 2.5-2.7 (2H, d, J=4H and t, J=4.5 Hz), 4.4-4.6 (1H, m),5.2 (2H, br. s), 5.44 and 5.67 (1H, 2s), 6.8-7.5 (20H, m).

Step D: Preparation of4-(Triphenylmethylthio)-1-[(benzyloxycarbonyl)triphenylphosphoranylidenemethyl]azetidin-2-one

A solution of4-(triphenylmethylthio)-1-(benzyloxycarbonylchloromethyl)azetidin-2-one(850 mg), triphenylphosphine (640 mg) and 2,6-butidine (210 μl) in DMF(50 ml) was heated at 80° C. under nitrogen for 24 hours. The reactionwas then cooled, diluted with ice water and extracted with ethyl acetate(2×100 ml). The ethyl acetate extracts were washed with water and brine,dried (Na₂ SO₄) and evaporated. The residue was flash chromatographedwith a solvent gradient of 15 to 60% ethyl acetate/hexane to affordrecovered starting material (500 mg) and then4-(triphenylmethylthio)-1-[(benzyloxycarbonyl)triphenylphosphoranylidenemethyl]azetidin-2-one(650 mg) as a white foam, R_(f) =0.20 (30% ethyl acetate/hexane).

Step E: Preparation of4-(Phenylcarbonylthio)-1-[(benzyloxycarbonyl)triphenylphosphoranylidenemethyl]azetidin-2-one

To a solution of4-(triphenylmethylthio)-1-[(benzyloxycarbonyl)triphenylphosphoranylidenemethyl]azetidin-2-one(200 mg) in methanol (5 ml) was added pyridine (25 μl ) followed bysilver nitrate (55 mg). After stirring at room temperature for 15minutes, the precipitate was collected by filtration, washed withmethanol and ether and dried in vacuo. The silver salt product (145 mg)was used directly.

The above silver salt (145 mg) was dissolved in methylene chloride (5 μl) and benzoyl chloride (41 μl ) were sequentially added. After 30 minthe reaction was poured into water and extracted with methylene chloride(2×). Each extract was washed with brine, dried (Na₂ SO₄) andevaporated. The residue was purified by preparative TLC (1×2000 μmsilica plates, 6% ethyl acetate/hexane) to afford4-(phenylcarbonylthio)-1-[(benzyloxycarbonyl)triphenylphosphoranylidenemethyl]azetidin-2-one(90 mg), R_(f) =0.32 (60% ethyl acetate/hexane).

Step F: Preparation of Benzyl 2-phenylpenem-3-carboxylate

A solution of4-(phenylcarbonylthio)-1-[(benzyloxycarbonyl)triphenylphosphoranylidenemethyl]azetidin-2-one(90 mg) in toluene (35 ml) was heated at 110°-120° C. under nitrogen for6 hours. The toluene was evaporated in vacuo and the residue waspurified by preparative TLC (2×1000 μm silica plates, 40% ethylacetate/hexane) to give the desired benzyl 2-phenylpenem-3-carboxylate(24 mg) as a white solid.

NMR δ(CDCl₃): 3.48 (1H dd, J=16 Hz, J=2 Hz), 3.80 (1H, dd, J=16 Hz,J=3.5 Hz), 5.07 (2H, s), 5.67 (1H, dd, J=3.5 Hz, J=2 Hz), 6.9-7.4 (10H,m).

EXAMPLE 2

Following the procedures described in Example 1, but starting with anappropriate 3-substituted-4-(acetoxy)azetidin-2-one as in Example 1,Step A and treating the resulting triphenylphosphoranylidenemethylintermediate with an appropriate carbonyl or thiocarbonyl chloride as inStep E, the following benzyl penem-3-carboxylates were prepared.

    ______________________________________                                         ##STR32##                                                                    R.sub.1     R              B                                                  ______________________________________                                        H           CH.sub.3       CH.sub.2 C.sub.6 H.sub.5                           H           CH.sub.2 C.sub.6 H.sub.5                                                                     CH.sub.2 C.sub.6 H.sub.5                           C.sub.2 H.sub.5                                                                           C.sub.6 H.sub.5                                                                              CH.sub.2 C.sub.6 H.sub.5                            .sub.-i-C.sub.3 H.sub.7                                                                  CH.sub.3       CH.sub.2 C.sub.6 H.sub.5                            .sub.-i-C.sub.3 H.sub.7                                                                  CH.sub.2 C.sub.6 H.sub.5                                                                     CH.sub.2 C.sub.6 H.sub.5                            .sub.-i-C.sub.3 H.sub.7                                                                  C.sub.6 H.sub.5                                                                              CH.sub.2 C.sub.6 H.sub.5                            .sub.-i-C.sub.3 H.sub.7                                                                   ##STR33##     CH.sub.2 C.sub.6 H.sub.5                            .sub.-i-C.sub.3 H.sub.7                                                                  SC.sub.2 H.sub.5                                                                             CH.sub.2 C.sub.6 H.sub.5                           CH.sub.3 CH(OH)                                                                           SC.sub.2 H.sub.5                                                                             CH.sub.2 (p-NO.sub.2 C.sub.6 H.sub.4)              ______________________________________                                    

EXAMPLE 3 Benzyl 2-methylpenem-3-carboxylate-1-sulfoxide

A solution of benzyl 2-methylpenem-3-carboxylate (160 mg) in methylenechloride (10 ml) was cooled to -20° C. and m-chloroperbenzoic acid (140mg) was added. The reaction was allowed to warm to room temperature over2 hours and then quenched into an aqueous solution of sodium bicarbonate(5%) and sodium sulfite. The product was extracted into methylenechloride (2×) and the extract were each washed with brine, dried (Na₂SO₄) and evaporated in vacuo. The residue was purified by preparativeTLC (1×2000 μm silica plate, 60% ethyl acetate/hexane) to give benzyl2-methylpenem-3-carboxylate-1-sulfoxide (100 mg) as a white solid.

NMR δ(CDCl₃): 2.50 (3H, s), 3.30 (1H, dd, J=5 Hz, J=16 Hz), 3.60 (1H,dd, 16 Hz, 30 Hz), 4.73 (1H, dd, 3 Hz, 5 Hz), 5.27 (2H, s), 7.3 (5H, br.s).

EXAMPLE 4 p-Nitrobenzyl2-(ethylthio)-6-[2-(benzyloxycarbonyloxy)ethyl]penem-3-carboxylate

To a solution of p-nitrobenzyl2-(ethylthio)-6-[(2-hydroxy)ethyl]penem-3-carboxylate (20 mg) inmethylene chloride (0.5 ml) at room temperature was added4-dimethylaminopyridine (12 μl) and benzyl chloroformate (14 μl). Flashchromatography (40% ethyl acetate/hexane) of the reaction mixturewithout work-up gave p-nitrobenzyl2-(ethylthio)-6-[2-(benzyloxycarbonyloxy)ethyl]penem-3-carboxylate (6mg).

NRM δ(CDCl₃): 1.33 (3H, t, J=7 Hz), 1.50 (3H, d, J=8 Hz), 2.98 (2H, m),3.90 (1H, m), 5.11 (2H, s), 5.3 (2H, ABq), 7.30 (5H, s), 6.78 (2H, d,J=9 Hz), 8.13 (2H, d, 9 Hz).

What is claimed is:
 1. A method of treating or managingelastase-mediated diseases comprising the administration to a mammalianspecies in need of such treatment an effective amount of a compound ofstructural formula: ##STR34## wherein M is:(1) --OR wherein R is asdefined below; (2) --SR; (3) --SOR; (4) --SO₂ R; (5) --COOR; (6) --OCOR;(7) phenyl; (8) --CH₂ A wherein A represents:(a) hydrogen; (b) C₁₋₆alkyl; (c) phenyl; (d) OR; (e) halo; (f) OCOR; (g) SR or S(CS)OR_(a)wherein R_(a) is C₁₋₆ alkyl or H; (h) SCOR; (i) CH₂ NHCHO; (j) CH₂ N₃ ;(k) CH₂ NH₂ ; or (l) CH₂ OH; (9) hydrogen; R is:(a) hydrogen; (b) C₁₋₆alkyl; (c) phenyl; (d) --CH₂ CH₂ NH₂ ; (e) --CH₂ CH₂ NHCOOCH₃ ; (f)--CH₂ CH₂ OCH₃ ; (g) tetrazolyl; or (h) C₆ H₅ CH₂ --; R₁ is:(1)hydrogen; (2) C₁₋₆ alkyl; (3) OR; (4) SR; (5) C₁₋₆ hydroxyalkyl; (6)C₁₋₆ alkoxycvasrbonyl-C₁₋₆ alkyl; (7) C₁₋₆ alkoxycarbonyloxy-C₁₋₆ alkyl;(8) benzoycarbonyloxy-C₁₋₆ alkyl; or (9) phenoxycarbonyloxy-C₁₋₆ alkyl;B is OB₁ or NB₂ B₃ wherein B₁ and B₂ independently are(1) benzyl; (2)phenyl; (3) straight or branched C₁₋₆ alkyl; (4) straight or branchedC₂₋₈ alkenyl; (5) C₃₋₈ cycloalkyl; (6) C₁₋₆ alkanoyloxy C₁₋₆ alkyl; (7)C₁₋₆ alkanoyl C₁₋₆ alkyl; (8) C₁₋₆ alkoxy C₁₋₆ alkyl; or (9) halo-C₁₋₆alkyl;the above groups (1)-(9) can optionally be substituted by radicalsselected from a group consisting of C₁₋₆ alkyl, hydroxy, C₁₋₆ alkoxy,halo, nitro, mercapto, amino, cyano, carboxy, sulfoamino, carbamoyl,carbamoyloxy C₁₋₆ alkyl or amino sulfonyl, C₁₋₆ alkylamino, sulfamoyl,azido, carboxamido or N-C₁₋₆ alkylcarboxamido: n is 0, 1 or
 2. 2. Themethod of claim 1 wherein:M is:(1) C_(l-6) alkyl; (2) phenyl; (3) SR;(4) OR; (5) CH₂ A wherein A is:(a) phenyl; (b) OH; (c) SR; or ##STR35##wherein R_(a) is H or C₁₋₆ alkyl; (d) OR; (e) OCOR_(c) wherein R_(c) isH, C₁₋₆ alkyl, phenyl, benzyl or C₁₋₆ alkylamino; or (f) hydrogen; Ris(1) C₁₋₆ alkyl; ##STR36## (3) --CH₂ --C₆ H₅ ; (4) phenyl; or (5)hydrogen; R₁ is(1) C₁₋₃ alkyl; (2) OR; (3) hydrogen; (4)benzoxycarbonyloxy-C₁₋₃ alkyl; (5) C₁₋₆ alkoxycarbonyloxy-C₁₋₃ alkyl; or(6) hydroxy-C₁₋₆ alkyl; B is OB₁ wherein B₁ is:(1) benzyl; (2) ethyl;(3) t-butyl; (4) --CH₂ CH₂ CH═CH₂ ; (5) --CH₂ CH₂ CH₂ COOt-Bu; (6) C₁₋₆alkanoyloxymethyl; or (7) C₁₋₆ alkanoylmethyl; n is 0 to
 1. 3. Themethod of claim 1 wherein the active compound is selected from a groupconsisting of compounds as defined below:

    __________________________________________________________________________     ##STR37##                                                                    R.sub.1        n  M           B                                               __________________________________________________________________________    H              0  CH.sub.3    OCH.sub.2 C.sub.6 H.sub.5                       H              1  CH.sub.3    OCH.sub.2 C.sub.6 H.sub.5                       (CH.sub.3).sub.2 CH                                                                          0  CH.sub.2 C.sub.6 H.sub.5                                                                  OCH.sub.2 C.sub.6 H.sub.5                       (CH.sub.3).sub.2 CH                                                                          0  C.sub.6 H.sub.5                                                                           OCH.sub.2 C.sub.6 H.sub.5                       CH.sub.3 O     0  CH.sub.2 OCOCH.sub.3                                                                      OCH.sub.2 C.sub.6 H.sub.5                       CH.sub.3 CH(OH)                                                                              0  SC.sub.2 H.sub.5                                                                          OCH.sub.2 (p-NO.sub.2 C.sub.6 H.sub.5)          CH.sub.3 CH(OCOOCH.sub.2 C.sub.6 H.sub.5)                                                    0  SC.sub.2 H.sub.5                                                                          OCH.sub.2 (p-NO.sub.2 C.sub.6 H.sub.5)          (CH.sub.3).sub.2 CH                                                                          0                                                                                 ##STR38##  OCH.sub.2 C.sub.6 H.sub.5                       H              0  OC.sub.2 H.sub.5                                                                          OCH.sub.2 C.sub.6 H.sub.5                       C.sub.2 H.sub.5                                                                              0  C.sub.6 H.sub.5                                                                           OCH.sub.2 C.sub.6 H.sub.5                       H              0  H           OCH.sub.2 C.sub.6 H.sub.5                       H              0  CH.sub.2 C.sub.6 H.sub.5                                                                  OCH.sub.2 C.sub.6 H.sub.5                       (CH.sub.3).sub.2 CH                                                                          0  CH.sub.3    OCH.sub.2 C.sub.6 H.sub.5                       __________________________________________________________________________


4. The method of treatment of claim 1 wherein the active compound isbenzyl 2-phenylpenem-3-carboxylate.
 5. The method of treatment of claim1 wherein the active compound is benzyl2-methylpenem-3-carboxylate-1-sulfoxide.
 6. The method of treatment ofclaim l wherein the active compound is p-nitrobenzyl2-ethylthio-6-[2-(benzyloxycarbonyloxy)penem-3]-carboxylate.